SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness

Results

When cultured under standard laboratory conditions in rich medium, null mutations of each of the three SOS pols have no observable effect on cellular growth (Fig. 1A). Individually cultured pol⁻ cells show the same cell yields after overnight growth and the same stationary-phase survival patterns as the wild-type. In fact, mutants of pol II, pol IV, and pol V can survive at least 2 months of stationary-phase incubation, without the addition of nutrients, at cell densities similar to the wild-type (data not shown). However, in an environment where wild-type and mutant strains are cocultured and must compete for nutrients, the situation changes dramatically. Each pol⁻ strain displays a significant reduction in relative fitness leading to its eventual extinction from the population when cocultured with the isogenic wild-type parent (Fig. 1B). Mutant cells deficient in pol II, pol IV, or pol V cannot survive more than 10 days of coculture with wild-type cells. Each mutant strain can survive for up to 5 days but then begins to disappear, decreasing at rates of up to an order of magnitude per day. After 10–14 days of incubation, no pol⁻ mutant cells remain in the culture. Double mutants of each pairwise combination of pol⁻ alleles, as well as the pol II⁻, pol IV⁻, and pol V⁻ triple mutant, show fitness losses during coculture more quickly than the single mutants (data not shown).

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Figure 1

Long-term survival patterns of wild-type and polymerase mutant cells. (A) Composite of survival patterns when cells are grown alone in batch culture. (B) Composite of survival patterns of each pol⁻ strain when grown in competition with wild-type cells. Representative growth curves are shown. Solid line with filled boxes, wild-type; dashed lines are mutant strains: open squares, pol II⁻; open circles, pol IV⁻; open triangles, pol V⁻ mutants. CFU/ml, colony-forming units per milliliter. Asterisks correspond to cell titers below the limit of detection of 100 CFU/ml.

We have verified that SOS polymerase genes are transcribed during stationary phase in the absence of exogenous DNA damage (Fig. 2). RT-PCR products corresponding to transcripts of all three SOS polymerase genes and recA have been obtained from cells incubated for 1–5 days. In addition, we have observed RT-PCR products of transcripts corresponding to the stationary-phase-specific gene dps (35), encoding a highly abundant, nonspecific DNA-binding protein, and sbmC, which encodes a gene regulating susceptibility to the peptide antibiotic microcin B17. sbmC is expressed both during stationary phase and upon SOS induction (36). Null mutants of each DNA polymerase strain do not yield an RT-PCR product (Fig. 2A).

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Figure 2

Transcription of the SOS-induced DNA polymerases. RT-PCR assays were performed on wild-type (WT), polB (pol II⁻), dinB (pol IV⁻), or umuDC (pol V⁻) cells harvested from 1-day-old cultures (A) or 5-day-old wild-type cultures (B). Gene names are indicated above each lane. The expected sizes of RT-PCR products are: sbmC, 311 bp; recA, 328 bp; umuD, 298 bp; dinB 206 bp, polB, 367 bp; dps, 209 bp.

Each pol⁻ mutant shows a competitive disadvantage when cocultured with the wild-type parent (Fig. 1B), yet the mutants show similar relative fitness when competing against each other. In this case, we do not mean to imply that the two competing pol⁻ (null mutant) populations remain at high density for the entire incubation period. Instead, we observe that when many initially identical cocultures are prepared, roughly half of the time, one pol⁻ strain dominates and half of the time the other mutant wins. This “flip-flop” in survival suggests that each pol⁻ mutant accumulates either deleterious or possibly advantageous mutations similarly, resulting in similar overall fitness profiles. We show a representative experiment in which two different polymerase null mutant strains, in this case mutants of pol IV and pol V, are cocultured and incubated under long-term stationary-phase conditions (Fig. 3). In each trial of the experiment, neither strain completely outcompetes the other. More pol V⁻ cells exist relative to pol IV⁻ cells at the conclusion of one trial (Fig. 3A), but more pol IV⁻ cells than pol V⁻ at the end of the second trial (Fig. 3B). In the third trial, although pol IV⁻ cells outnumber pol V⁻ cells at the end of the experiment, the pol V⁻ cells are increasing in relative numbers (Fig. 3C). Several iterations of these experiments with all three possible pairs of competing strains (pol II⁻ vs. pol IV⁻, pol II⁻ vs. pol V⁻, and pol IV⁻ vs. pol V⁻) showed no clear winners or losers. None of the pol⁻ strains displayed an overall competitive advantage or disadvantage versus other pol⁻ strains, whereas their lack of competitive fitness was obvious when growing in the presence of wild-type cells (Fig. 1B).

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Figure 3

Representative competitions between polymerase-deficient strains. Solid lines with open squares, pol IV⁻; dashed lines with open circles, pol V⁻. CFU/ml, colony forming units per milliliter.

Long thought of as a period of stasis, the stationary phase of the bacterial life cycle during batch culture incubation is now known to be a highly dynamic period during which many genetic systems are induced that ensure the long-term survival of the population (30, 32). As incubation continues in the absence of new nutrients, further cellular changes occur eventually leading to the death of more than 99% of cells. It is from the survivors of this “death phase” that another change soon becomes apparent. Among this population emerge cells expressing the Growth Advantage in Stationary Phase, or GASP, phenotype (28, 29). GASP results from the appearance and selection of advantageous mutations that confer a competitive fitness advantage on a subset of cells within the population during stationary phase.

Previous data (Fig. 1) demonstrate that each SOS polymerase confers an important advantage for survival on a short-term basis. We now address whether these polymerases also play a role in the long-term survival of bacteria in stationary phase during nutrient stress. In other words, do SOS polymerase mutants exhibit the GASP phenotype as do their wild-type parents?

In a typical GASP competition experiment, cells from a 10-day-old wild-type culture are inoculated as a numerical minority (1:1,000 vol/vol) into a fresh (1-day-old) culture. Both subpopulations are monitored for the next few weeks, and their relative proportion in the population is determined. When wild-type cells are aged for 10 days, they invariably display the GASP phenotype, completely outcompeting the parental nonaged strain after 10–12 days of coculture (Fig. 4, class 1). The results are dramatically different when the GASP analysis is performed by using SOS polymerase mutants (Fig. 4). Individual cultures of wild-type or pol⁻ strains were cultured for 10 days in LB. After 10 days, a 5-μl sample of each culture was inoculated directly into 1-day-old 5-ml cultures of the wild-type parent. Wild-type and pol⁻ mutant strains carry unique antibiotic resistance markers allowing each to be distinguished. Unlike wild-type cells that display a strong GASP phenotype (class 1) after 10 days of incubation, in virtually every case, the pol⁻ mutant strains display up to four different phenotypes or “flavors” of GASP.

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Figure 4

GASP competition phenotypes of wild-type and polymerase mutant strains during coculture. Competition phenotypes are defined as: class 1, strong GASP phenotype; class 2, weaker GASP phenotype; class 3, abortive expression of GASP; class 4, no expression of the GASP phenotype. Representative examples of each class are shown. Black lines represent titers of unaged populations; gray lines represent titers of cells obtained from 10-day-old aged cultures. The frequency, in percentage, of expression of each class of GASP competition phenotype for wild-type and mutant strains is shown. n = 14 for wild-type (WT), pol II⁻, and pol IV⁻ strains; n = 18 for pol V⁻ strains. CFU/ml, colony forming units per milliliter.

In addition to the class 1 competition, where aged cells completely outcompete nonaged cells, we also observed competition in which aged cells were able to increase their relative frequency in the population displaying equal fitness to the nonaged wild-type parent (class 2). A third type of competition (class 3) is observed where the aged cells attempt, but fail, to outcompete the wild-type. These pol mutants are less fit, because, after initially expressing a growth advantage, increasing their relative frequency in the population for a short time, they are completely displaced by nonaged parental cells. A last competition class is observed (class 4) where aged mutant strains are completely unable to compete with the nonaged wild-type parent. Previous studies of the ability of wild-type cells to express the GASP phenotype (28–32) revealed only two competition classes (equivalent to classes 1 and 4 described here.) This study identifies two new classes of GASP (classes 2 and 3) in which the potential competitive advantages gained by the acquisition of new alleles are countered by the competitive fitness loads of carrying mutant pol alleles.

Because of the disparate fidelity properties of the three SOS polymerases, it might be expected that the acquisition of mutations conferring a competitive advantage during stationary-phase growth may differ depending on the pol mutant allele. It seems that pol V may play a more significant role in the acquisition of the GASP phenotype, whereas pol II and IV mutants acquire a GASP phenotype about 86 and 79% of the time, respectively (including both class 1 and 2 competitions), pol V mutant strains GASP only 44% of the time. Although the pol V mutants express the GASP phenotype less frequently than the other two mutant strains, when the pol V⁻ cells do express a competitive advantage, it is usually of the stronger phenotypic class (class 1). In other words, aged pol V⁻ strains are very good competitors almost as frequently as they are very poor competitors (Fig. 4).

Although SOS polymerase-deficient strains do not always express the GASP phenotype when competing with their wild-type parent, they do express GASP when competing with other pol⁻ strains. In almost every case, aged 10-day-old pol⁻ cells, when inoculated as a minority into a young culture, express the GASP phenotype, outcompeting the nonaged (1-day-old) mutant strains in the majority (data not shown). The one exception was for GASP competitions between aged pol V⁻ mutant strains and nonaged pol IV⁻ strains. In these experiments, pol V mutants failed to express the GASP phenotype in two of three trials (data not shown). The interesting question raised by these results is whether mutations that appear during long-term incubation that do not confer the GASP phenotype are simply not the “right” kind of mutations to allow expression of a competitive advantage or whether the abundance of any beneficial mutation is countered by the load of a reduction in replication efficiency caused by carrying a pol⁻ phenotype.

Discussion

When looking only at large populations of isogenic cells growing in batch culture, the types of fitness defects reported here are impossible to detect because the cells are indistinguishable: they all carry the same genetic markers. If an individual cell suffers a deleterious mutation, it is replaced by the progeny of viable siblings. Although growth rates seem to be normal and populations seem to be maintained during long-term incubation, these cultures are actually highly dynamic. GASP mutants with increased fitness appear in “waves” that can sweep through the culture as it ages (28, 29, 31, 32). Cells that are hobbled in their ability to grow, even to a relatively modest degree, will over time be out-competed by more fit cells. When the types of DNA lesions that induce the SOS polymerases are encountered, replication is impeded, causing a loss in competitive fitness. Each SOS polymerase may make its own specialized contribution to maintaining fitness and the generation of genetic diversity by filling in for a temporarily indisposed replicative pol III core polymerase during error-free replication-fork restart (pol II), putative error-prone replication fork rescue (pol IV), or error-prone translesion synthesis (primarily pol V.) Pols II and V also contribute to error-prone and error-free translesion synthesis depending on the specific identity of the lesion encountered (1, 22).

The relative fitness of any SOS polymerase mutant growing under the conditions of a GASP competition experiment reflects a balance between opposing selective forces: the appearance of positively selected advantageous mutations versus the negative effects of reduced replication efficiency. The competition data shown in Fig. 1B reflect the fitness load placed on each of the pol mutant strains in a standard competition experiment. These fitness reductions may reflect the inability of these cells to replicate efficiently during long-term stationary-phase incubation. However, we do not believe that the reduced fitness phenotype during GASP competition is wholly attributable to reductions in replication efficiency. The observation that all three pol mutant strains are able to express a class 1 GASP competition phenotype (Fig. 4) suggests that the role of reduction in replication can be outweighed by a cell's ability to generate and maintain new advantageous alleles. Yet, the contribution of a loss in replication efficiency to this reduced fitness of SOS polymerase mutants remains an open question.

The observation that pol V mutants show the greatest reduction in class 1 GASP competitions suggests that a subset of mutations, attributable especially to pol V, may provide the mutational “raw material” on which natural selection acts during the evolution of bacterial populations, in a manner superficially similar to the increased fitness accompanying the absence of MutSLH mismatch repair system (37–40). A key distinction to be drawn between the kinds of mutational events associated with the loss of MutSLH mismatch repair and those associated with the loss of SOS polymerases is that replication persists, under most conditions, whether or not the mismatch repair system is functioning, allowing the propagation of missense mutations. When the types of DNA lesions that induce the repair polymerases are encountered, replication often halts, with dire consequences for the cell. The observation that the polB, dinB, and umuDC genes (along with known stationary-phase-specific genes, dps and sbmC) are transcribed during stationary phase in the absence of induction of the SOS system by exogenous DNA-damage agents suggests that the activities of these polymerases are essential for long-term survival. Given the observation that all three SOS pols can perform in both an error-prone or error-free manner, beyond the repair of DNA damage, these data support a model in which these alternative DNA polymerase enzymes are important for the generation of genetic diversity under normal physiological conditions.